CN105044334A - Surface plasma resonance method for detecting peanut allergen - Google Patents
Surface plasma resonance method for detecting peanut allergen Download PDFInfo
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Abstract
The invention discloses a surface plasma resonance method for detecting a peanut allergen. The method specifically comprises the steps: respectively preparing an anti-peanut Ara h1 monoclonal antibody and an anti-peanut Ara h1 polyclonal antibody, fixing the anti-peanut Ara h1 monoclonal antibody on a detection chip by means of a coupling method, facilitating peanut Ara h1 proteins of different concentrations to flow by the detection chip, adding the anti-peanut Ara h1 polyclonal antibody which is used for magnifying a signal, so that a purpose of rapidly and efficiently detecting a main allergen protein of the peanut can be achieved. The method has characteristics of high sensitivity, high accuracy, rapidness in detection and the like and can become an important technical means for detecting the peanut allergen.
Description
Technical field
The present invention relates to technical field of immunoassay, particularly relate to a kind of Surface Plasmon Resonance detecting Peanut Allergen.
Background technology
What FAO/WHO assert causes in eight of human foods allergy large based foods, and peanut and goods thereof are exactly one of them.In the food labelling method of America and Europe, also specify peanut is the food hypersenstivity ultimate constituent that must indicate.Arah1 albumen is the main anaphylactogen in peanut.
The detection method of Peanut Allergen mainly for the DNA fragmentation of specific allergy crude protein in peanut or coding anaphylactogen for detecting target.Therefore, detection method is mainly divided into two classes: to detect method based on gene and to detect the method based on albumen.Rely on PCR (PCR) to increase specific DNA fragmentation by the method detected based on gene, this class methods difinite quality PCR and real-time fluorescence quantitative PCR (Real-timePCR) etc.Qualitative PCR mainly detects by the agarose electrophoresis of mixing fluorescent dye the specific DNA whether having peanut.Better DNA detection method is exactly Real-timePCR, and specific probe can be utilized to improve the specificity detected.Also have RT-PCR method in addition, this method needs strict experimental implementation, commercially can not get widespread use.Detect the total protein of peanut or special Peanut Allergen by the method detected based on albumen, these class methods generally include immunoassay detection method, as DI(DotImmunoblotting), RAST (Radio-allergosorbenttest), RIE(Rocketimmuno-electrophoresis), ELISA(Enzyme-linkedimmunosorbentassay), DSA(Dipstickassay) and LFIA (lateralflowimmunoassay) etc.Wherein, DI, DSA and LFIA are quantitative and semi-quantitative methods, and DSA completes whole process by continuous print three step, 30min to 3h consuming time; And LFIA completes by means of only a step, about 5min to 10min consuming time; RIE needs electrophoresis and immuning hybridization, operates consuming time, and the later stage can not get widely using.RAST and ELISA is quantitative analysis method.ELISA is high and easy to operate because of its accuracy, commonly uses in general food is analyzed.These methods depend on specificity and the sensitivity of antibody used.
The Peanut Allergen composition detection of current commercialization mainly contains three kinds of method: ELISA, colloidal gold method (LFIA) and quantitative real-time PCR.The method of protein detection of ELISA and LFIA mainly detects peanut total protein in food or peanut main allergen (Arah1 or Arah2), and the detection rising limit (LOD) of ELISA is generally slightly generally be about 5ppm lower than the LOD of 5ppm, LFIA.The about 10min that detection time about 2h, the LFIA of ELISA are used.Compared with LFIA, ELISA is more sensitive, but needs the more running time; Compared with ELISA, LFIA does not need extra detecting instrument, more simple and easy, fast, and cost-saving.But LFIA only can provide the testing result of Yes/No, and ELISA can sxemiquantitative or complete quantitatively detection.Commercial real-time fluorescence quantitative PCR reagent mainly detects peanut main allergic protogene (Arah2) DNA level in food, and need 1h, LOD is about 10-50ppm.
Therefore, prior art has yet to be improved and developed.
Summary of the invention
In view of above-mentioned the deficiencies in the prior art, the object of the present invention is to provide a kind of Surface Plasmon Resonance detecting Peanut Allergen, be intended to solve that existing detection method exists takes time and effort, the not high and problem that Allergic skin test does not go out or cost is too high to low dosage of sensitivity.
Technical scheme of the present invention is as follows:
Detect a Surface Plasmon Resonance for Peanut Allergen, wherein, comprise step:
A, prepare peanut Arah1 albumen;
B, using peanut Arah1 albumen as antigen, immune animal, prepares anti-peanut Arah1 polyclonal antibody;
C, using peanut Arah1 albumen as antigen, immune animal, obtain hybridoma, prepare anti-peanut Arah1 monoclonal antibody by hybridoma;
D, by the method for coupling, anti-peanut Arah1 monoclonal antibody is fixed in detection chip, by the peanut Arah1 protein stream of variable concentrations through described detection chip, then adds anti-peanut Arah1 polyclonal antibody for amplifying signal, obtain testing result.
The Surface Plasmon Resonance of described detection Peanut Allergen, wherein, described steps A specifically comprises: carry out the process of acetone degrease to peanut, then by PBS damping fluid, peanut protein extraction is carried out to the peanut after degrease, obtain peanut protein crude extract, isolate the peanut Arah1 albumen in peanut protein crude extract finally by ammonium sulfate precipitation and anion exchange chromatography.
The Surface Plasmon Resonance of described detection Peanut Allergen, wherein, described step B specifically comprises: using peanut Arah1 albumen as antigen, by immune animal after peanut Arah1 albumen and Freund's complete adjuvant mixing and emulsifying, simultaneously by immune animal after peanut Arah1 albumen and freund 's incomplete adjuvant mixing and emulsifying, then press from both sides animal eyeball and get blood, centrifuging and taking serum, obtained anti-peanut Arah1 polyclonal antibody.
The Surface Plasmon Resonance of described detection Peanut Allergen, wherein, also comprises after described step B: antagonism peanut Arah1 polyclonal antibody carries out titration, purifying and specific assay.
The Surface Plasmon Resonance of described detection Peanut Allergen, wherein, described step C specifically comprises: using peanut Arah1 albumen as antigen, by immune animal after peanut Arah1 albumen and Freund's complete adjuvant mixing and emulsifying, simultaneously by immune animal after peanut Arah1 albumen and freund 's incomplete adjuvant mixing and emulsifying, then get the animal splenocyte after immunity to mix with myeloma cell, Fusion of Cells is carried out by PEG method, obtain hybridoma, carry out screening to hybridoma and clone, obtained anti-peanut Arah1 monoclonal antibody.
The Surface Plasmon Resonance of described detection Peanut Allergen, wherein, described step C also comprises antagonism peanut Arah1 monoclonal antibody and carries out purification process, described purification process specifically comprises: be target antigen with peanut Arah1 albumen, application ELISA method is carried out cross matching and is got rid of nonspecific reaction, get positive hybridoma cell strain brine 3 times, be made into 1 × 106/mL, inoculation every, animal abdominal cavity 0.5mL, ascites is extracted after 8 ~ 10d, centrifugal, be separated ascites, packing, purifying ascites, obtain two strains anti-peanut Arah1 monoclonal antibody, called after 2G9 and 5G4 respectively.
The Surface Plasmon Resonance of described detection Peanut Allergen, wherein, described step C also comprises employing Western blot antagonism peanut Arah1 monoclonal antibody to be identified, and adopts different anti-igg type monoclonal antibody antagonism Arah1 monoclonal antibodies to carry out Antibody types mensuration.
The Surface Plasmon Resonance of described detection Peanut Allergen, wherein, described step D specifically comprises step:
D1, anti-peanut Arah1 monoclonal antibody is fixed in detection chip;
D2, with the flow velocity of 3 ~ 7 μ L/min injection peanut Arah1 protein stream chip 280 ~ 320s after testing;
D3, with the flow velocity of 3 ~ 7 μ L/min injection HBS-EP damping fluid balance detection chip surface 150 ~ 200s;
D4, inject anti-peanut Arah1 polyclonal antibody for amplifying signal with the flow velocity of 3 ~ 7 μ L/min;
D5, with the flow velocity of 25 ~ 35 μ L/min injection 8 ~ 10mMGlycine-HCl25 ~ 35s, 3 surfaces for anti-peanut Arah1 monoclonal antibody;
Before non-zero concentration injection, substitute peanut Arah1 albumen with HBS-EP damping fluid and be used for detecting LOD as sample zero-dose.
The Surface Plasmon Resonance of described detection Peanut Allergen, is characterized in that, in described step D1, the fixing of described anti-peanut Arah1 monoclonal antibody specifically comprises step:
D11, injection HBS-EP damping fluid, with the flow velocity balance detection chip surface 4 ~ 7min of 8 ~ 12 μ L/min;
The mixed liquor of D12, injection NHS and EDC, with the flow velocity of 8 ~ 12 μ L/min activation detection chip surface 5 ~ 10min;
D13, inject anti-peanut Arah1 monoclonal antibody, with the flow velocity coupling detection chip 5 ~ 10min of 8 ~ 12 μ L/min;
D14, injection monoethanolamine, close detection chip surface 5 ~ 10min with the flow velocity of 8 ~ 12 μ L/min.
The Surface Plasmon Resonance of described detection Peanut Allergen, wherein, in described step D2, be 5 with HBS-EP damping fluid by the dilution of peanut Arah1 albumen, 10,20,40,80,100,500,1000,4000,16000,50000, the peanut Arah1 albumen of the variable concentrations of 100000ng/mL.
Beneficial effect: the present invention utilizes anti-peanut Arah1 monoclonal antibody as sessile antibody, catches and grabs peanut Arah1 albumen, then carries out signal amplification by anti-peanut Arah1 polyclonal antibody, reaches the object rapidly and efficiently carrying out detecting peanut major allergen protein.The inventive method combines specificity and the SPR feature rapidly and efficiently of double-antibodies sandwich ELISA simultaneously, thus reach efficiently, accurately, Detection results easily.
Embodiment
The invention provides a kind of Surface Plasmon Resonance detecting Peanut Allergen, for making object of the present invention, technical scheme and effect clearly, clearly, the present invention is described in more detail below.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
The invention provides a kind of Surface Plasmon Resonance detecting Peanut Allergen, it comprises step:
A, prepare peanut Arah1 albumen;
B, using peanut Arah1 albumen as antigen, immune animal, prepares anti-peanut Arah1 polyclonal antibody;
C, using peanut Arah1 albumen as antigen, immune animal, obtain hybridoma, prepare anti-peanut Arah1 monoclonal antibody by hybridoma;
D, by the method for coupling, anti-peanut Arah1 monoclonal antibody is fixed in detection chip, by the peanut Arah1 protein stream of variable concentrations through described detection chip, then adds anti-peanut Arah1 polyclonal antibody for amplifying signal, obtain testing result.
Surface plasma resonance (SPR) method is based on optical technology principle by analyzing molecules interphase interaction information, and can detect the reaction between one or more molecules in real time, be a kind of novel foodstuff Allergic skin test method.This SPR method is without background interference, lower to the degree of purification of sample, use simple, fast, without the need to mark, highly sensitive.
The present invention utilizes SPR method to detect peanut main allergen peanut Arah1 albumen.Specifically prepare anti-peanut Arah1 monoclonal antibody and anti-peanut Arah1 polyclonal antibody respectively, then by the method for coupling, anti-peanut Arah1 monoclonal antibody is fixed in detection chip, by the peanut Arah1 protein stream chip after testing of variable concentrations, add anti-peanut Arah1 polyclonal antibody for amplifying signal, thus reach the object rapidly and efficiently detecting peanut major allergen protein.The inventive method have highly sensitive, accuracy is high, detect the features such as quick.
Below by a specific embodiment, technique scheme of the present invention is described in detail.
One, the preparation of peanut Arah1 albumen
1, acetone degrease
Pulverize with the natural peanut of Philip soy bean milk making machine by 200g drying, then peanut powder is placed in the beaker of 500mL, add proper amount of acetone, magnetic agitation two hours in 4 DEG C of refrigerators, repeated multiple times degrease, till supernatant clarification.Then remove acetone, in fuming cupboard, make acetone air-dry in degrease peanut powder, after air-dry, obtain defatted peanut powder.
2, slightly the carrying of peanut protein
Weigh peanut powder, add PBS damping fluid by every gram of sample 7mL and carry out protein extraction, 4 DEG C of magnetic agitation 24h.Carried rear refrigerated centrifuge at 15000r/min, centrifugal 15min under 4 DEG C of conditions, supernatant is Peanut Allergen crude extract.
3, ammonium sulfate precipitation
Peanut Allergen crude extract is placed in beaker, slowly adds ammonium sulfate until saturation degree reaches 30% when magnetic agitation, treat to dissolve completely, by crude extract centrifugal 10min of 12000r/min at 4 DEG C.Precipitation is dissolved completely with appropriate PBS; By identical operation make centrifugal after the ammonium sulfate concentrations of supernatant reach 70%, third time reaches 100%.The protein solution of ammonium sulfate three fractional precipitation gained is preserved in 4 DEG C of refrigerators.
4, anion exchange chromatography is separated peanut Arah1 albumen
The preparation of level pad, elution buffer: level pad contains 8M urea, the Tris-HCl(pH8.0 of 20mM).Elution buffer is identical with equalizing and buffering liquid making method, except the NaCl adding 600mM.
Cross the preparation of chromatographic column protein sample: first by the level pad of the protein sample 1mL mixing 3mL of ammonium sulfate precipitation, ultrafiltration is centrifugal in the MILLIPORE ultra-filtration centrifuge tube of 15mL, the centrifugal 10min of 5000r/min.Go filtrate, then add level pad 3mL, ultrafiltration is centrifugal, 5 times so repeatedly.Make protein sample solution similar to level pad, final sample volume is 1mL.
In the full-automatic chromatograph system of KTApurifier (GE company), strong anion displacement chromatography MonoQ5/50GL is adopted to carry out separation and purification of protein, after peanut protein upper prop, with elution buffer with 0 ~ 600mM/NaCl continuous gradient wash-out, 280nm detects ultraviolet absorption peak, collects each eluting peak.
5, SDS-PAGE electrophoretic analysis
Adopt discontinuous system SDS-PAGE electrophoresis slab gel, concentrated gum concentration is 5%, and resolving gel concentration is 12%, first uses 80V constant voltage electrophoresis 15min, then 120V constant voltage leakage of electricity swimming.Through coomassie brilliant blue R250 dyeing, acetic acid decolouring after electrophoresis terminates.
6, the mensuration of peanut Arah1 protein concentration
Adopt BCA method, with BCA protein content kit, make the standard peanut Arah1 protein solution of variable concentrations.Measure the light absorption value of variable concentrations standard peanut Arah1 protein solution respectively at 562nm place with ultraviolet spectrophotometer, with absorbance (A562) for ordinate, standard protein content (ug/uL) is horizontal ordinate, drawing standard curve.
7, Western blot qualification peanut Arah1 albumen
The purifying protein liquid getting 10uL carries out SDS-PAGE electrophoresis, the complete rear taking-up SDS-PAGE gel of electrophoresis, by the size clip nitrocellulose filter (NC film) of gel, then under 350mA condition, and transferring film 50min in 4 DEG C of refrigerators.Take off NC film 3%BSA in 4 DEG C of refrigerators close spend the night, add irritated patient positive pooled serum (by 1:10 dilution).After shaking table shakes 2h, NC film is proceeded in biotin labeling sheep antihuman IgE antibody (by 1:1500 dilution) and shake 2h.Streptavidin (HRP) wherein shakes 2h by being put into by NC film after 1:1000 dilution.Last DAB colour developing, namely rinse with water after band is clear, color development stopping is reacted.。
Two, anti-peanut Arah1 polyclonal antibody preparation
1, first immunisation
After getting 100 μ g peanut Arah1 albumen and isopyknic Freund's complete adjuvant mixing and emulsifying, hypodermic injection mouse carotid back.Booster immunization totally 2 times, protein content is the same, is mixed into the freund 's incomplete adjuvant of equivalent.Each immunization interval about 3 weeks, after the 3rd immunity, the 5th day, takes folder eyeball to get blood, centrifuging and taking serum.Qualification for anti-peanut Arah1 polyclonal antibody have employed Western blot (Western-blotting) qualification, first carry out SDS-PAGE electrophoresis with the peanut Arah1 albumen extracted, then electricity forwards on nitrocellulose filter, be primary antibodie with immunized mice serum, with mice serum before immunity for negative control, be two to resist with the antibody of sheep anti-mouse igg of mark HRP, develop the color with DAB.
2, the titration of anti-peanut Arah1 polyclonal antibody and purifying
Conventional indirect elisa method is adopted to detect, the peanut Arah1 albumen extracted is spent the night by 4 DEG C, 100ng/ hole bag, and confining liquid is 2%BSA-PBST, 37 DEG C of closed 2h, with the polyvalent antibody obtained for primary antibodie, do parallel negative control experiments, two-fold dilution from 1:100 with mice serum before immunity, 37 DEG C of reaction 1h simultaneously, after PBST washes three times, add against murine IgG-HRP to resist as two, 37 DEG C of reaction 1h, add substrate TMB and develop the color after washing three times.Measure hole OD value and be greater than 2.1 for positive criterion with the ratio of negative control hole OD value, the dilutability of antibody is tiring of antibody.Identify and after titration, adopt folder eyeball to get blood, centrifuging and taking serum, for serum antibody purifying.Antibody purification carries out with reference to HiTrapproteinG affinity chromatography instructions.
3. the specific assay of anti-peanut Arah1 polyclonal antibody
Can detecting this anti-peanut Arah1 polyclonal antibody with indirect ELISA some nut fruitses of recognition (almond, English walnut, walnut, fibert and American pistachios), the albumen such as some beans product (soybean, broad bean, pea, black soya bean, mung bean, red bean) and sunflower seed, wheat, buckwheat, rye, egg, milk, sesame, shrimp, fish.Spent the night by 4 DEG C, 100ng albumen/hole bag in every hole, confining liquid is 2%BSA-PBST, 37 DEG C of closed 2h, and with this anti-peanut Arah1 polyclonal antibody for primary antibodie, be positive control with peanut Arah1 albumen, take PBS as negative control simultaneously.
Three, anti-peanut Arah1 monoclonal antibody preparation
1, the preparation of anti-peanut Arah1 monoclonal antibody and its purifying
First by peanut Arah1 protein immunization BALB/C mice after purifying, specific requirement is according to the animal immune in how anti-preparation process, then the splenocyte of the BALB/C mice after purifying peanut Arah1 protein immunization is taken, by with NS-1 rat bone marrow tumour cell in 2: 1 ratio mix, carry out Fusion of Cells by PEG method.Be added in 96 orifice plates of existing feeder layer, put 37 DEG C, 5%CO
2cultivate in incubator.HAT selects to cultivate, and filters out required hybridoma cell line, after positive aperture 4 subclones, chooses last clone and expands cultivation, produce antibody.By indirect immunoperoxidase mark method screening positive clone and subgroup identification.Be target antigen with purifying peanut Arah1 albumen, application ELISA method is carried out cross matching and is got rid of nonspecific reaction.Get well-grown hybridoma cell strain brine 3 times, be made into 1 × 106/mL, every, Mice Inoculated abdominal cavity 0.5mL, extracts ascites after 8 ~ 10d, centrifugal, and be separated ascites, packing ,-20 DEG C save backup.The affine displacement chromatography post of ProteinG (GE company) instructions of the purifying reference antibody purification of monoclonal antibody in ascites.Mainly obtain the anti-Arah1 antibody of two strains, respectively called after 2G9 and 5G4.
The mensuration of 2, the qualification of anti-peanut Arah1 monoclonal antibody, Antibody types and antibody titer
Qualification for anti-peanut Arah1 monoclonal antibody have employed Western blot (Western-blotting) qualification, first carry out SDS-PAGE electrophoresis with the peanut Arah1 albumen extracted, then electricity forwards on nitrocellulose filter, be primary antibodie by the anti-peanut Arah1 monoclonal antibody after purifying, with mice serum before immunity for negative control, be two to resist with the antibody of sheep anti-mouse igg of mark HRP, colour developing.Different anti-igg type monoclonal antibodies is adopted to carry out Antibody types mensuration to the anti-peanut Arah1 monoclonal antibody after purifying.Conventional indirect elisa method is adopted to detect, natural Peanut Allergen albumin A rah1 is spent the night by 4 DEG C, 100ng/ hole bag, and confining liquid is 3%BSA-PBST, 37 DEG C of closed 2h, with the anti-peanut Arah1 monoclonal antibody after purifying for primary antibodie, do parallel negative control experiments, two-fold dilution from 1:100 with mice serum before immunity, 37 DEG C of reaction 1h simultaneously, after PBST washes three times, add against murine IgG-HRP to resist as two, 37 DEG C of reaction 1h, add substrate TMB and develop the color after washing three times.Measure hole OD value and be greater than 2.1 for positive criterion with the ratio of negative control hole OD value, the dilutability of antibody is tiring of antibody.
3, the specific assay of anti-peanut Arah1 monoclonal antibody
Can detecting this two strain anti-peanut Arah1 monoclonal antibody (2G9 and 5G4) with indirect ELISA some nut fruitses of recognition (almond, English walnut, walnut, fibert and American pistachios), the albumen such as some beans product (soybean, broad bean, pea, black soya bean, mung bean, red bean) and sunflower seed, wheat, buckwheat, rye, egg, milk, sesame, shrimp, fish.Spent the night by 4 DEG C, 100ng albumen/hole bag in every hole, confining liquid is 2%BSA-PBST, 37 DEG C of closed 2h, and with this antibody for primary antibodie, be positive control with peanut protein, take PBS as negative control simultaneously.
Four, SPR detects
This experiment uses SPR method (GEHealthcare, BIAcoreT200) to detect.
Material
CM5 chip, Cat.No.BR-1005-30 (GEHealthcare);
NHS:100mMN-N-Hydroxysuccinimide;
EDC:400mM dimethylaminopropyl ethyl phosphoamide;
HBS-EP damping fluid;
Monoethanolamine: 1M monoethanolamine, pH8.5;
Purifying peanut Arah1 albumen: purity >90%, 1.0mg/mL, is kept in PBS damping fluid;
Purifying anti-peanut Arah1 monoclonal antibody 5G4: purity >90%, 1.8mg/mL, is kept in PBS damping fluid;
Purifying anti-peanut Arah1 polyclonal antibody: purity >90%, 1.0mg/mL, is kept in PBS damping fluid;
Method
Purifying anti-peanut Arah1 monoclonal antibody 5G4's is fixing
By the method for amino coupled, anti-for purifying peanut Arah1 monoclonal antibody 5G4 is fixed on CM5 chip:
Surface balance: HBS-EP damping fluid, with the flow velocity of 10 μ L/min balance chip surface 5min;
Surface active: injection " NHS+EDC " 1:1 mixed liquor, with the flow velocity of 10 μ L/min activation chip surface 7min;
Coupling: injection purifying anti-peanut Arah1 monoclonal antibody 5G4(is diluted in 10mM sodium acetate, in pH4.5 damping fluid), with the flow velocity coupling 7min of 10 μ L/min;
Surface-closed: injection monoethanolamine, with the flow velocity confining surface 7min of 10 μ L/min.
Antibody sensitivity technique
Be 5 with HBS-EP damping fluid by the dilution of purifying peanut Arah1 albumen, 10,20,40,80,100,500,1000,4000,16000,50000,100000ng/mL detects, all concentration all repeats following process:
Certain density purifying peanut Arah1 albumen 300s is injected with the flow velocity of 5 μ L/min;
With the flow velocity of 5 μ L/min injection HBS-EP damping fluid 180s balance surface;
Amplifying signal is used for the flow velocity of 5 μ L/min injection 0.3mg/mL purifying anti-peanut Arah1 polyclonal antibody;
With the flow velocity of 30 μ L/min injection 10mMGlycine-HCl(pH2.0) 30s, 3 times for regenerating the surface of purifying anti-peanut Arah1 monoclonal antibody 5G4.
Before non-zero concentration injection, substitute certain density purifying peanut Arah1 albumen with HBS-EP damping fluid and be used for detecting LOD(and lowest detectable limit as sample zero-dose (n=5): the least concentration of the checking matter that can detect under background signal; Computing method are three times of sample zero-dose signal averaging SD).
Detected the method for peanut main allergen Arah1 albumen by the above-mentioned SPR set up, the LOD of anti-peanut Arah1 monoclonal antibody is 2.58ng/mL; The LOD of anti-peanut Arah1 polyclonal antibody is 0.77ng/mL, compared to additive method, the maximum advantage of the inventive method is without the need to marking sample, easy to detect, quick, highly sensitive and can repeatedly utilize, the amount that sample needs is few, can high flux, analyze data in high quality.
The present invention utilizes the principle of the single-minded identification of antigen-antibody, in conjunction with SPR efficiently, accurately and rapidly the main allergen Arah1 albumen of advantage to peanut detect.Compared with prior art, core improvements of the present invention are to pass through SPR technique, utilize the anti-peanut Arah1 monoclonal antibody of high-titer as sessile antibody, catch and grab peanut Arah1 albumen, carry out signal amplification by anti-peanut Arah1 polyclonal antibody again, rapidly and efficiently carry out detection peanut major allergen protein.This method combines specificity and the SPR feature rapidly and efficiently of double-antibodies sandwich ELISA simultaneously, thus reach efficiently, accurately, Detection results easily.
Should be understood that, application of the present invention is not limited to above-mentioned citing, for those of ordinary skills, can be improved according to the above description or convert, and all these improve and convert the protection domain that all should belong to claims of the present invention.
Claims (10)
1. detect a Surface Plasmon Resonance for Peanut Allergen, it is characterized in that, comprise step:
A, prepare peanut Arah1 albumen;
B, using peanut Arah1 albumen as antigen, immune animal, prepares anti-peanut Arah1 polyclonal antibody;
C, using peanut Arah1 albumen as antigen, immune animal, obtain hybridoma, prepare anti-peanut Arah1 monoclonal antibody by hybridoma;
D, by the method for coupling, anti-peanut Arah1 monoclonal antibody is fixed in detection chip, by the peanut Arah1 protein stream of variable concentrations through described detection chip, then adds anti-peanut Arah1 polyclonal antibody for amplifying signal, obtain testing result.
2. the Surface Plasmon Resonance of detection Peanut Allergen according to claim 1, it is characterized in that, described steps A specifically comprises: carry out the process of acetone degrease to peanut, then by PBS damping fluid, peanut protein extraction is carried out to the peanut after degrease, obtain peanut protein crude extract, isolate the peanut Arah1 albumen in peanut protein crude extract finally by ammonium sulfate precipitation and anion exchange chromatography.
3. the Surface Plasmon Resonance of detection Peanut Allergen according to claim 1, it is characterized in that, described step B specifically comprises: using peanut Arah1 albumen as antigen, by immune animal after peanut Arah1 albumen and Freund's complete adjuvant mixing and emulsifying, simultaneously by immune animal after peanut Arah1 albumen and freund 's incomplete adjuvant mixing and emulsifying, then press from both sides animal eyeball and get blood, centrifuging and taking serum, obtained anti-peanut Arah1 polyclonal antibody.
4. the Surface Plasmon Resonance of detection Peanut Allergen according to claim 1, is characterized in that, also comprises after described step B: antagonism peanut Arah1 polyclonal antibody carries out titration, purifying and specific assay.
5. the Surface Plasmon Resonance of detection Peanut Allergen according to claim 1, it is characterized in that, described step C specifically comprises: using peanut Arah1 albumen as antigen, by immune animal after peanut Arah1 albumen and Freund's complete adjuvant mixing and emulsifying, simultaneously by immune animal after peanut Arah1 albumen and freund 's incomplete adjuvant mixing and emulsifying, then get the animal splenocyte after immunity to mix with myeloma cell, Fusion of Cells is carried out by PEG method, obtain hybridoma, carry out screening to hybridoma and clone, obtained anti-peanut Arah1 monoclonal antibody.
6. the Surface Plasmon Resonance of detection Peanut Allergen according to claim 1, it is characterized in that, described step C also comprises antagonism peanut Arah1 monoclonal antibody and carries out purification process, described purification process specifically comprises: be target antigen with peanut Arah1 albumen, application ELISA method is carried out cross matching and is got rid of nonspecific reaction, get positive hybridoma cell strain brine 3 times, be made into 1 × 106/mL, inoculation every, animal abdominal cavity 0.5mL, ascites is extracted after 8 ~ 10d, centrifugal, be separated ascites, packing, purifying ascites, obtain two strains anti-peanut Arah1 monoclonal antibody.
7. the Surface Plasmon Resonance of detection Peanut Allergen according to claim 1, it is characterized in that, described step C also comprises employing Western blot antagonism peanut Arah1 monoclonal antibody to be identified, and adopts different anti-igg type monoclonal antibody antagonism peanut Arah1 monoclonal antibodies to carry out Antibody types mensuration.
8. the Surface Plasmon Resonance of detection Peanut Allergen according to claim 1, is characterized in that, described step D specifically comprises step:
D1, anti-peanut Arah1 monoclonal antibody is fixed in detection chip;
D2, with the flow velocity of 3 ~ 7 μ L/min injection peanut Arah1 protein stream chip 280 ~ 320s after testing;
D3, with the flow velocity of 3 ~ 7 μ L/min injection HBS-EP damping fluid balance detection chip surface 150 ~ 200s;
D4, inject anti-peanut Arah1 polyclonal antibody for amplifying signal with the flow velocity of 3 ~ 7 μ L/min;
D5, with the flow velocity of 25 ~ 35 μ L/min injection 8 ~ 10mMGlycine-HCl25 ~ 35s, 3 surfaces for anti-peanut Arah1 monoclonal antibody;
Before non-zero concentration injection, substitute peanut Arah1 albumen with HBS-EP damping fluid and be used for detecting LOD as sample zero-dose.
9. the Surface Plasmon Resonance of detection Peanut Allergen according to claim 8, is characterized in that, in described step D1, the fixing of described anti-peanut Arah1 monoclonal antibody specifically comprises step:
D11, injection HBS-EP damping fluid, with the flow velocity balance detection chip surface 4 ~ 7min of 8 ~ 12 μ L/min;
The mixed liquor of D12, injection NHS and EDC, with the flow velocity of 8 ~ 12 μ L/min activation detection chip surface 5 ~ 10min;
D13, inject anti-peanut Arah1 monoclonal antibody, with the flow velocity coupling detection chip 5 ~ 10min of 8 ~ 12 μ L/min;
D14, injection monoethanolamine, close detection chip surface 5 ~ 10min with the flow velocity of 8 ~ 12 μ L/min.
10. the Surface Plasmon Resonance of detection Peanut Allergen according to claim 8, it is characterized in that, in described step D2, be 5 with HBS-EP damping fluid by the dilution of peanut Arah1 albumen, 10,20,40,80,100,500,1000,4000,16000,50000, the peanut Arah1 albumen of the variable concentrations of 100000ng/mL.
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| CN106918707A (en) * | 2015-12-25 | 2017-07-04 | 广州瑞博奥生物科技有限公司 | A kind of antibody chip kit for detecting cell adhesion molecule |
| CN107345958A (en) * | 2017-06-23 | 2017-11-14 | 浙江诺迦生物科技有限公司 | The detection method of trace methamphetamine and the SPR chips used in a kind of saliva sample |
| CN108530513A (en) * | 2018-04-16 | 2018-09-14 | 河北农业大学 | Wheat flour anaphylactogen high efficiency extraction agent and its application method |
| CN111909938A (en) * | 2020-07-14 | 2020-11-10 | 深圳大学 | Peanut mutant gene, protein coded by same and preparation method of peanut mutant |
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| CN106918707A (en) * | 2015-12-25 | 2017-07-04 | 广州瑞博奥生物科技有限公司 | A kind of antibody chip kit for detecting cell adhesion molecule |
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| CN107345958A (en) * | 2017-06-23 | 2017-11-14 | 浙江诺迦生物科技有限公司 | The detection method of trace methamphetamine and the SPR chips used in a kind of saliva sample |
| CN108530513A (en) * | 2018-04-16 | 2018-09-14 | 河北农业大学 | Wheat flour anaphylactogen high efficiency extraction agent and its application method |
| CN108530513B (en) * | 2018-04-16 | 2021-08-17 | 河北农业大学 | Efficient wheat flour allergen extracting agent and using method thereof |
| CN111909938A (en) * | 2020-07-14 | 2020-11-10 | 深圳大学 | Peanut mutant gene, protein coded by same and preparation method of peanut mutant |
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